Add time:09/05/2019         Source:sciencedirect.com

An ultra high pressure liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method for the simultaneous quantitation of pravastatin and major metabolites, 3′α-hydroxy-pravastatin, pravalactone and 3′α-hydroxy-pravalactone, in human plasma has been developed and validated. Aliquots of (100 μL) plasma in EDTA were diluted in pH 4.5 (0.1 M buffer) to stabilize the analytes and subjected to hydrophilic lipophilic balance (HLB) solid phase extraction on 96 well μelution plates. Extracted samples were evaporated to dryness and reconstituted in pH 4.5 buffer. Chromatographic separation was performed on a Cortecs™ C18 column (2.1 × 100 mm, 1.8 μm), using gradient elution with a blend of acetonitrile and 10 mM methylammonium acetate buffer (pH 4.5) at a flow rate of 0.4 mL/min. Mass spectrometric detection was performed using multiple reaction monitoring (MRM) switching between positive/negative electrospay ionization (ESI). Pravastatin, 3′α-hydroxy-pravastatin, and internal standards [2H3]-pravastatin, and [2H3]-3′α-hydroxy-pravastatin were monitored in negative ESI mode at ion transitions m/z 423.2 → 321.1 and 426.2 → 321.1, respectively. Positive ESI mode was used for the detection of pravalactone, 3′α-hydroxy-pravalactone, and internal standards [2H3]-pravalactone, and [2H3]-3′α-hydroxy-pravalactone at ion transitions m/z 438.2 → 183.1 and 441.2 → 269.1 respectively. The method was linear for all analytes in the concentration range 0.5–200 nM with intra- and inter-day precisions (as relative standard deviation) of ≤5.2% and accuracy (as relative error) of ≤8.0% at all quality control levels. The method was successfully applied to the investigation of pharmacokinetic properties of pravastatin and its metabolites in children after an oral dose of 20–40 mg.

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